Recombinant protein: production methods and applications

Science 2023

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Recombinant protein: production methods and applications
Recombinant protein: production methods and applications
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Protein is an essential component of all organisms. Each of its molecules consists of one or more polypeptide chains consisting of amino acids. Although the information necessary for life is encoded in DNA or RNA, recombinant proteins perform a wide range of biological functions in organisms, including enzymatic catalysis, protection, support, movement, and regulation. According to their functions in the body, these substances can be divided into different categories, such as antibodies, enzymes, structural component. Given their important functions, such compounds have been intensively studied and widely used.

lab expression

In the past, the main way to obtain a recombinant protein was to isolate it from a natural source, which is usually inefficient and time consuming. Recent advances in biological molecular technology have made it possible to clone DNA encoding a specific set of substances into an expression vector for substances such as bacteria, yeast, insect cells, and mammalian cells.

Simply put, recombinant proteins are translated by exogenous DNA products intoliving cells. Getting them usually involves two main steps:

  1. Cloning a molecule.
  2. Protein expression.

Currently, the production of such a structure is one of the most powerful methods used in medicine and biology. The composition has a wide application in research and biotechnology.

Medical direction

Recombinant proteins provide important treatments for various diseases such as diabetes, cancer, infectious diseases, hemophilia and anemia. Typical formulations of such substances include antibodies, hormones, interleukins, enzymes, and anticoagulants. There is a growing need for recombinant formulations for therapeutic use. They allow you to expand treatment methods.

genetically engineered recombinant proteins play a key role in the therapeutic drug market. Mammalian cells currently produce the most therapeutic agents because their formulations are capable of producing high quality, natural-like substances. In addition, many approved recombinant therapeutic proteins are produced in E. coli due to good genetics, rapid growth, and high productivity. It also has a positive effect on the development of drugs based on this substance.

Research

Obtaining recombinant proteins is based on different methods. Substances help to find out the basic and fundamental principles of the body. These molecules can be used to identify and determinelocation of the substance encoded by a particular gene, and to reveal the function of other genes in various cellular activities such as cell signaling, metabolism, growth, replication and death, transcription, translation and modification of the compounds discussed in the article.

Modern methods of obtaining

Thus, the observed composition is often used in molecular biology, cell biology, biochemistry, structural and biophysical studies and many other fields of science. At the same time, obtaining recombinant proteins is an international practice.

Such compounds are useful tools in understanding intercellular interactions. They have proven effective in several laboratory methods such as ELISA and immunohistochemistry (IHC). Recombinant proteins can be used to develop enzyme assays. When used in combination with a pair of appropriate antibodies, the cells can be used as standards for new technologies.

Biotechnology

Recombinant proteins containing an amino acid sequence are also used in industry, food production, agriculture and bioengineering. For example, in animal husbandry, enzymes can be added to food to increase the nutritional value of feed ingredients, reduce costs and waste, support animal gut he alth, improve productivity, and improve the environment.

genetic editing

In addition, lactic acid bacteria (LAB) for a long timehave been used to produce fermented foods, and recently LAB has been developed for the expression of recombinant proteins containing an amino acid sequence, which can be widely used, for example, to improve human, animal and nutritional digestion.

However, these substances also have limitations:

  1. In some cases, the production of recombinant proteins is complex, costly and time consuming.
  2. Substances produced in cells may not match natural forms. This difference can reduce the effectiveness of therapeutic recombinant proteins and even cause side effects. In addition, this difference may affect the results of experiments.
  3. The main problem with all recombinant drugs is immunogenicity. All biotech products can exhibit some form of immunogenicity. It is difficult to predict the safety of new therapeutic proteins.

In general, advances in biotechnology have increased and facilitated the production of recombinant proteins for a variety of applications. Although they still have some drawbacks, the substances are important in medicine, research and biotechnology.

Disease link

recombinant protein is not harmful to humans. It is only an integral part of the overall molecule in the development of a specific drug or nutritional element. Many medical studies have shown that forced expression of the FGFBP3 protein (abbreviated BP3) in a laboratory strain of obese mice showed a significant reduction in their body fat.mass, despite the genetic predisposition to use.

The results of these trials show that the FGFBP3 protein may offer a new therapy for disorders associated with metabolic syndrome such as type 2 diabetes and fatty liver disease. But because BP3 is a natural protein and not an artificial drug, clinical trials of recombinant human BP3 could begin after the final round of preclinical studies. On, that is, there are reasons related to the safety of conducting such studies. The recombinant protein is not harmful to humans due to its stepwise processing and purification. Changes are taking place at the molecular level as well.

PD-L2, one of the key players in immunotherapy, was nominated for the 2018 Nobel Prize in Physiology or Medicine. This work, started by Prof. James P. Allison from the USA and Prof. Tasuku Honjo from Japan, has led to the treatment of cancers such as melanoma, lung cancer, and others based on checkpoint immunotherapy. Recently, AMSBIO has added a major new product to its immunotherapy line, the PD-L2/TCR activator - CHO Recombinant Cell Line.

In proof-of-concept experiments, researchers at the University of Alabama at Birmingham, led by H. Long Zheng, MD, Professor Robert B. Adams, and Director of Laboratory Medicine, Department of Pathology, UAB School of Medicine, have highlighted a potential therapy a rare but fatal bleeding disorder, TTP.

The results of thisstudies demonstrate for the first time that transfusion of rADAMTS13-loaded platelets may be a novel and potentially effective therapeutic approach for arterial thrombosis associated with congenital and immune-mediated TTP.

Recombinant protein is not only a nutrient, but also a drug in the composition of the drug being developed. These are just a few areas that are now involved in medicine and related to the study of all its structural elements. As international practice shows, the structure of a substance makes it possible at the molecular level to deal with many serious problems in the human body.

Vaccine development

A recombinant protein is a specific set of molecules that can be modeled. A similar property is used in the development of vaccines. A new vaccination strategy, also known as the use of a special recombinant virus injection, could protect millions of chickens at risk from a serious respiratory illness, researchers from the University of Edinburgh and the Pirbright Institute said. These vaccines use harmless or weak versions of a virus or bacterium to introduce germs into the body's cells. In this case, experts used recombinant viruses with different spike proteins as vaccines to create two versions of a harmless virus. There are many different drugs built around this connection.

New approach to treatment

Recombinant protein trade names and analogues are as follows:

  1. "Fortelizin".
  2. "Z altrap".
  3. "Eylea".

These are mainly anticancer drugs, but there are other areas of treatment associated with this active substance.

A new vaccine, also called LASSARAB, designed to protect people against both Lassa fever and rabies, has shown promising results in preclinical studies, according to a new study published in the scientific journal Nature Communications. An inactivated recombinant vaccine candidate uses a weakened rabies virus.

The research team inserted Lassa virus genetic material into a rabies virus vector so that the vaccine would express surface proteins in both Lassa and rabies cells. These surface compounds elicit an immune response against infectious agents. This vaccine was then inactivated to "destroy" the live rabies virus used to make the carrier.

Getting Methods

There are several systems for producing a substance. The general method for obtaining a recombinant protein is based on obtaining biological material from the synthesis. But there are other ways.

Currently there are five main expression systems:

  1. E. coli expression system.
  2. Yeast expression system.
  3. Insect cell expression system.
  4. Mammalian cell expression system.
  5. Cell-free protein expression system.

The latter option is particularly suitable for the expression of transmembrane proteinsand toxic compounds. In recent years, substances that are difficult to express by conventional intracellular methods have been successfully integrated into cells in vitro. In Belarus, the production of recombinant proteins is widely used. There are a number of state-owned enterprises dealing with this issue.

Cell Free Protein Synthesis System is a fast and efficient method for synthesizing target substances by adding various substrates and energy compounds necessary for transcription and translation in the enzymatic system of cellular extracts. In recent years, the advantages of cell-free methods for types of substances such as complex, toxic membranes have gradually emerged, demonstrating their potential application in the biopharmaceutical field.

Cell-free technology can add a variety of non-naturally occurring amino acids easily and in a controlled manner to achieve complex modification processes that are difficult to resolve after conventional recombinant expression. Such methods have high application value and potential for drug delivery and vaccine development using virus-like particles. A large number of membrane proteins have been successfully expressed in free cells.

Expression of compositions

Recombinant protein CFP10-ESAT 6 is produced and used to create vaccines. Such a tuberculosis allergen allows you to strengthen the immune system and develop antibodies. In general, molecular studies involve the study of any aspect of a protein, such as structure, function, modifications, localization, or interactions. To explorehow specific substances regulate internal processes, researchers usually require the means to produce functional compounds of interest and benefit.

Creating Vaccines

Given the size and complexity of proteins, chemical synthesis is not a viable option for this endeavor. Instead, living cells and their cellular machinery are usually used as factories to create and construct substances based on the provided genetic templates. The recombinant protein expression system then develops the necessary structure to create a drug. Next comes the selection of the necessary material for different categories of drugs.

Unlike proteins, DNA is easy to construct synthetically or in vitro using well-established recombinant techniques. Therefore, DNA templates of specific genes, with or without added reporter sequences or affinity tag sequences, can be designed as templates for expression of the monitored substance. Such compounds derived from such DNA templates are called recombinant proteins.

Traditional strategies for expression of a substance involve transfecting cells with a DNA vector that contains a template and then culturing the cells to transcribe and translate the desired protein. Typically, the cells are then lysed to extract the expressed compound for subsequent purification. The recombinant protein CFP10-ESAT6 is processed in this way and goes through a purification system from possiblethe formation of toxins. Only after that it goes to be synthesized into a vaccine.

Both prokaryotic and eukaryotic in vivo expression systems for molecular substances are widely used. The choice of system depends on the type of protein, the requirement for functional activity, and the desired yield. These expression systems include mammals, insects, yeasts, bacteria, algae and cells. Each system has its own advantages and challenges, and choosing the right system for a particular application is important for the successful expression of the substance under review.

Expression from mammals

The use of recombinant proteins allows the development of vaccines and drugs of various levels. For this, this method of obtaining a substance can be used. Mammalian expression systems can be used to produce proteins from the animal kingdom that have the most native structure and activity due to their physiologically relevant environment. This results in high levels of post-translational processing and functional activity. Mammalian expression systems can be used to produce antibodies, complex proteins, and compounds for use in cell-based functional assays. However, these benefits are coupled with more stringent culture conditions.

Mammalian expression systems can be used to generate proteins transiently or through stable cell lines where the expression construct is integrated into the host genome. While such systems can be used in multiple experiments, the timeproduction can generate a large amount of substance in one to two weeks. This type of recombinant protein biotechnology is in high demand.

These transient, high-yielding mammalian expression systems use suspension cultures and can yield grams per litre. In addition, these proteins have more native folding and post-translational modifications such as glycosylation compared to other expression systems.

Insect Expression

Methods for producing recombinant protein are not limited to mammals. There are also more productive ways in terms of production costs, although the yield of the substance per 1 liter of treated liquid is much lower.

Clinical Trials

Insect cells can be used to express a high level protein with modifications similar to mammalian systems. There are several systems that can be used to generate recombinant baculovirus, which can then be used to extract the substance of interest in insect cells.

Expressions of recombinant proteins can be easily scaled up and adapted to high density suspension culture for large-scale compounding of molecules. They are more functionally similar to the native composition of mammalian matter. Although the yield can be up to 500 mg/L, the production of recombinant baculovirus can be time consuming and culture conditions are more difficult than prokaryotic systems. However, in more southern and warmer countries, a similarmethod is considered more efficient.

Bacterial expression

Production of recombinant proteins can be established with the help of bacteria. This technology is much different from those described above. Bacterial protein expression systems are popular because the bacteria are easy to culture, grow quickly, and give high yields of the recombinant formulation. However, multidomain eukaryotic substances expressed in bacteria are often non-functional because cells are not equipped to perform the necessary post-translational modifications or molecular folding.

In addition, many proteins become insoluble as inclusion molecules, which are very difficult to recover without harsh denaturators and subsequent cumbersome molecular refolding procedures. This method is mostly considered to be still largely experimental.

Cell free expression

Recombinant protein containing the amino acid sequence of staphylokinase is obtained in a slightly different way. It is included in many types of injections, requiring several systems before use.

Cell free protein expression is an in vitro synthesis of a substance using translationally compatible whole cell extracts. In principle, whole cell extracts contain all the macromolecules and components required for transcription, translation, and even post-translational modification.

These components include RNA polymerase, regulatory protein factors, transcription forms, ribosomes and tRNA. When addingcofactors, nucleotides and a specific gene template, these extracts can synthesize proteins of interest in a few hours.

Although not sustainable for large-scale production, cell-free or in vitro protein expression (IVT) systems offer a number of advantages over conventional in vivo systems.

Cell-free expression allows rapid synthesis of recombinant formulations without involving cell culture. Cell-free systems make it possible to label proteins with modified amino acids, as well as to express compounds that undergo rapid proteolytic degradation by intracellular proteases. In addition, it is easier to express many different proteins at the same time using a cell-free method (for example, testing protein mutations by small-scale expression from many different recombinant DNA templates). In this representative experiment, the IVT system was used to express the human caspase-3 protein.

Conclusions and future prospects

Recombinant protein production can now be seen as a mature discipline. This is the result of numerous incremental improvements in purification and analysis. Currently, drug discovery programs are rarely stopped due to the inability to produce the target protein. Parallel processes for the expression, purification and analysis of several recombinant substances are now well known in many laboratories around the world.

natural ingredients

Protein complexes and growing success in makingsolubilized membrane structures will require more changes to keep up with demand. The emergence of effective contract research organizations for a more regular supply of proteins will allow the reallocation of scientific resources to meet these new challenges.

Additionally, parallel workflows should allow the creation of complete libraries of the monitored substance to enable new target identification and advanced screening, along with traditional small molecule drug discovery projects.

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